Decorin Mimic Inhibits Vascular Smooth Muscle Proliferation and Migration

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.     

Apelin-13促进血管平滑肌细胞增殖、迁移和血管新生内膜增生

研究apelin-13对血管平滑肌细胞（vascular smooth muscle cell, VSMC）增殖和迁移的影响及其作用机制.用免疫印迹分析检测apelin-13对VSMC增殖、迁移以及分化相关基因表达的影响,结果表明,apelin-13能以时间和浓度依赖的方式诱导VSMC增殖和迁移相关基因cyclin D1和MMP-2表达,促进细胞增殖和迁移;同时使VSMC分化标志基因SM22α和SM α-actin表达水平降低.而且,用鬼笔环肽对细胞骨架进行染色的结果显示,apelin-13可以促进VSMC从收缩表型向增殖表型转化.体内实验也表明,敲低apelin可抑制球囊损伤诱导的新生内膜形成,提示apelin-13在体内具有促进血管新生内膜形成的作用.总之,本文结果表明,apelin 13通过调节VSMC增殖、迁移以及分化基因表达,进而促进其从分化型向增殖型转化,并向内膜下迁移和增殖.     

Interference of IP-10 Expression Inhibits Vascular Smooth Muscle Cell Proliferation and Intimal Hyperplasia in Carotid Artery: A New Insight in the Prevention of Restenosis

After vascular angioplasty, vascular smooth muscle cell (VSMC) proliferation causes atherosclerosis and intimal hyperplasia leading to restenosis. Interferon-γ-inducible protein (IP)-10 plays a role in atherogenesis, but the mechanism remains unclear. We evaluated the role of IP-10 in intimal hyperplasia and restenosis. IP-10 expression was determined in arterial specimens from 20 arteriosclerotic obliteration patients and 6 healthy individuals. VSMCs were stimulated in vitro with IFN-γ and transfected with IP-10 siRNA. Silencing was verified with RT-PCR/Western blot; cell proliferation rate was detected by methyl-thiazol-tetrazolium. The carotid artery model of atherosclerosis injury was established with IP-10 siRNA. IP-10 expression was detected at 1 and 4 weeks using RT-PCR and immunohistochemistry. Artery morphology was assessed with hematoxylin-and-eosin staining, and intimal hyperplasia was evaluated by electron microscopy. IP-10 was overexpressed in arteriosclerotic obliteration group compared with control group (P < 0.05). IP-10 expression in transfected group was significantly lower than in untransfected group. The intima-to-media ratio of transfected group at 4 weeks was lower than that of untransfected group (P < 0.01). The transfected group exhibited more regular intimal structure and less hyperplasia under electron microscopy. We, therefore, concluded that IP-10 played an important role in intimal hyperplasia as siRNA-mediated IP-10 silencing inhibited aberrant VSMCs hyperplasia and reduced restenosis.     

脂多糖对血管平滑肌细胞一氧化氮合酶基因表达及细胞增殖的影响

应用ＲＮＡ印迹分析和亚硝酸盐含量测定检查脂多糖（ＬＰＳ）对大鼠血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）基因表达及ＮＯ合成的影响,用３Ｈ－ＴｄＲ参入实验观察ＬＰＳ对细胞ＤＮＡ合成的影响．结果表明,ＬＰＳ在诱导ＶＳＭＣｉＮＯＳｍＲＮＡ表达和促进ＮＯ合成的同时,抑制ＶＳＭＣＤＮＡ合成．证明ＬＰＳ的作用与其浓度和作用时间有关     

miR-599 Inhibits Vascular Smooth Muscle Cells Proliferation and Migration by Targeting TGFB2

Aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of cardiovascular diseases including coronary heart disease, restenosis and atherosclerosis. MicroRNAs are a class of small, non-coding and endogenous RNAs that play critical roles in VSMCs function. In this study, we showed that PDGF-bb, as a stimulant, promoted VSMCs proliferation and suppressed the expression of miR-599. Moreover, overexpression of miR-599 inhibited VSMCs proliferation and also suppressed the PCNA and ki-67 expression. In addition, we demonstrated that ectopic expression of miR-599 repressed the VSMCs migration. We also showed that miR-599 inhibited type I collagen, type V collagen and proteoglycan expression. Furthermore, we identified TGFb2 as a direct target gene of miR-599 in VSMCs. Overexpression of TGFb2 reversed miR-599-induced inhibition of VSMCs proliferation and type I collagen, type V collagen and proteoglycan expression. In conclusion, our findings suggest miR-599 plays a crucial role in controlling VSMCs proliferation and matrix gene expression by regulating TGFb2 expression.     

Inhibition of Vascular Smooth Muscle Cell Proliferation by Gentiana lutea Root Extracts

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.     

Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor''s ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ), indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.     

Sodium Tanshinone IIA Silate Inhibits High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration through Activation of AMP-Activated Protein Kinase

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G₀/G₁ phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.     

糖尿病血管平滑肌细胞增殖与线粒体关系的研究进展

糖尿病(diabetes mellitus,DM)是严重危害人类健康的全身性代谢疾病,常发生血液流变学、微循环和细胞代谢的紊乱,导致缺血、缺氧和组织水肿,进而引起血管和神经病变。血管病变常常是糖尿病病人致死、致残的主要原因。血管平滑肌细胞的增殖及表型改变是糖尿病引发动脉粥样硬化性疾病的显著特征,而线粒体在血管平滑肌细胞的增殖过程中起重要作用。因此,探讨糖尿病血管平滑肌细胞与线粒体的关系及机制为糖尿病血管性疾病的治疗提供重要的理论基础,有利于基础研究向临床试用药物研究的转化。     

番茄红素对血管平滑肌细胞增殖与凋亡的影响

目的:VSMCs增殖是动脉粥样硬化的主要病理过程之一.本研究通过观察番茄红素对人血管平滑肌细胞(VSMC)增殖和凋亡的影响,并探讨与Toll样受体4(TLR-4)有关的信号通路相关分子机制,旨在为番茄红素治疗动脉粥样硬化提供理论基础.方法:番茄红素处理体外培养的VSMC,分别应用MTT及流式细胞术分析处理后的VSMC增殖及凋亡情况;采用荧光定量PCR和Western blot检测处理后VSMC的TLR-4及骨髓分化分子88 (MyD88)表达水平的变化;ELISA检测番茄红素对VSMC肿瘤坏死因子α(TNF-α)及白细胞介素6 (IL-6)分泌量的影响.结果:番茄红素处理后VSMC细胞增殖速度降低,凋亡增加;番茄红素处理后VSMC内TLR-4及MyD88的表达降低,TNF-α及IL-6分泌减少.结论:番茄红素可抑制VSMC生长增殖;促进凋亡,从而发挥治疗动脉粥样硬化的作用,其机制可能与TLR-4及MyD88表达降低、TNF-α及IL-6分泌减少有关.     

Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Critical Role of Exogenous Nitric Oxide in ROCK Activity in Vascular Smooth Muscle Cells

Objective

Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo.

Methods

VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor.

Results

Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP.

Conclusions

These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.     

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.     

同源异型盒基因对血管平滑肌细胞的调控作用

同源异型盒基因是一类对生物体的生长、发育和分化从时间和空间上进行协调的调控基因。构成血管中膜的血管平滑肌细胞表型具有极大的可塑性。在一些病理性血管重构时,血管平滑肌细胞可发生表型调变,从分化型调变为去分化型,具备增殖和迁移能力。在此过程中,多种同源异型盒基因的表达发挥了重要的调控作用。现就同源异型盒基因与血管平滑肌细胞的表型调变、增殖和迁移的关系等方面的研究进展作一综述。     

Evening Primrose Extracts Inhibit PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Regulating Cell-Cycle-Related Proteins

The proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors in the occurrence of cardiovascular diseases, such as blood flow abnormalities, stroke and atherosclerosis. Evening primrose, known as Oenothera biennis, is a plant native to Korea that exerts physiological activities, such as antioxidant effects, the inhibition of lipid accumulation and the prevention of muscle atrophy. However, the function of evening primrose stem (EVP) in the regulation of VSMC proliferation and migration and the underlying mechanisms have not been identified. In this study, the effect of EVP on the platelet-derived growth factor (PDGF)-induced proliferation and migration of VSMCs was investigated. The results show that PDGF-BB-induced proliferation of VSMCs was inhibited by EVP at concentrations of 25, 50 or 100 μg/mL in a concentration-dependent manner, and a migration assay showed that EVP inhibited cell migration. Cell cycle analysis was performed to confirm the mechanism by which cell proliferation and migration was inhibited. The results indicate that proteins involved in the cell cycle, such as cyclin, CDK and phosphorylated Rb, were downregulated by EVP at concentrations of 100 μg/mL, thereby increasing the proportion of cells in the G0/G1 phase and inhibiting cell cycle progression. In the PDGF receptor (PDGFR) signaling pathway, phosphorylation of the PDGFR was inhibited by EVP at concentrations of 100 μg/mL, and PLCγ phosphorylation was also decreased. The PDGF-BB-induced effect of EVP on the proliferation of VSMCs involved the inhibition of Akt phosphorylation and the reduction in the phosphorylation of MAPK proteins such as ERK, P38 and JNK. In conclusion, the results demonstrate that EVP inhibited PDGF-BB-induced VSMC proliferation and migration by regulating cell-cycle-related proteins.